Within recent years, plant genes and regulation thereof have been the object of intensive investigations. The plant genomes are complex and contain a large number of chromosomes which makes the elucidation of genes and especially the regulation thereof very difficult. Also, many plant genes are expressed at different times in the plant development providing differentiation of the plant cells.
.alpha.-Amylase, which is involved in the hydrolysis of starch, is present in all plants. .alpha.-Amylase in monocotyledonous plants such as barley and wheat has been extensively investigated and .alpha.-amylase genes from these monocotyledonous plants have been isolated. .alpha.-amylase from dicotyledonous plants has also been investigated, but only at the enzymatic level. It has not been possible to isolate or characterize .alpha.-amylase genes from dicotyledonous plants.
The present invention concerns DNA fragments related to the encoding of .alpha.-amylase in dicotyledonous plants and to important developments based on the provision of such DNA fragments.
The DNA fragments according to the invention and variants thereof, e.g., an .alpha.-amylase encoding part of a nucleotide sequence shown in FIG. 1-5, as well as subsequences and analogues thereof constitute the basis for transgenic plant strategies for modifying various essential properties of plants, e.g. of the genus Solanum tuberosum, such as deliberately decreasing .alpha.-amylase activity to reduce the risk of Maillard reactions and taste modification/degradations in connection with the production of potato chips etc., or for deliberately increasing .alpha.-amylase activity in potatoes which are to be used for fermentation for the production of spirits. These strategies, as well as other economically important transgenic plant strategies resulting in modification of the properties of plants made possible through the present invention are explained in greater detail below.
Besides their very important use for direct consumption, potatoes are used for many different industrial purposes such as in a raw material in the production of potato chips and in the production of alcohol. In both types of production, the degradation of starch in the potatoes is of importance and thus, .alpha.-amylase involved in the degradation of starch is an important enzyme, as explained below.
In the production of potato chips, the degradation of starch to reducing sugars is critical. A high degree of starch degradation resulting in a high content of reducing sugars causes a problem, since reducing sugar is subjected to the so-called Maillard reactions during the frying of the potatoes in vegetable oil, which causes the chips to acquire an undesirable brown colour and an unpleasant burned taste, accompanied by undesirable alterations in the flavour and texture of the potato chips.
On the other hand, a high content of reducing sugar resulting from a high .alpha.-amylase activity is desirable when potates are to be used for the production of alcohol.
For reference purposes, a very brief discussion of the use of potatoes for the production of chips and alcohol, respectively, is given below:
The production of potato chips starts with the peeling of the potatoes in special peeling machines. Next, the potatoes are sorted, whereby unfit potatoes, for instance green tubers or potatoes which have been damaged during harvesting, are removed. The potatoes then move on the sliding machines, where they are cut into slices of the appropriate thickness, typically about 1.5 mm. The sliced potatoes are then washed and drained, after which the potato slices are led to a large fryer, which typical has a capacity of about 2000 kg of potato chips per hour. After frying in the hot vegetable oil in the fryer, the potato slices emerge as finished potato chips and are ready for packaging. As mentioned above, undesired Maillard reactions will take place during the frying if reducing sugars are present in the potato slices.
For the production of alcohol from potatoes, the process starts with the boiling of the washed potatoes. The boiling gelatinizes the starch. The potatoes are typically boiled in an autoclave of a temperature of about 150.degree. C. or by use of a continuous process. During the heating to boiling, a thermolabile .alpha.-amylase, which has been added to the potato mass, is responsible for an initial saccharification in which an initial conversion of starch to reducing sugars is obtained. After boiling, the gelatinized starch is saccharified by the action of enzymes, whereby the viscous mash is liquified and the starch is converted to fermentable compounds such as glucose. The mash is cooled from a temperature of about 100.degree. C. to about 30.degree. C. before the addition of the enzymes. The enzymes are typically either enzymes extracted from green malt or dry malt (germinated barley) or e.g. bacterial .alpha.-amylase or fungal amyloglucosidase. Yeast is added to the mash after saccharification, and the fermentation of the fermentable sugars to alcohol and carbon dioxide takes place over a period of 2-3 days. The alcohol is then removed from the mash by continuous distillation, producing a crude alcohol of about 95% by volume, which can be further purified by rectification.
Starch exists in two different forms, amylose and amylopectin. Amylose, which is an unbranched form of starch, consists of D-glucose units connected by .alpha.-1,4 linkages. Amylopectin, which is a branched form of starch, comprises D-glucose units joined by .alpha.-1,6 linkages as well as .alpha.-1,4 linkages, the ratio between .alpha.-1,6 and .alpha.-1,4 linkages being on the order of about 1 to 30. It is believed that the branched amylopectins contain about 2000 to about 200,000 glucose units, while the unbranched amylose molecules contain a few thousand glucose units.
Both amylopectin and amylose are hydrolyzed by .alpha.-amylase, which hydrolyzes the internal .alpha.-1,4 linkages of yield maltose, which consists of two glucose units, maltotriose, which consists of three glucose units, and .alpha.-dextrin, which consists of several glucose units comprising an .alpha.-1,6 linkages and .alpha.-1,4 linkages. The products of hydrolysis by .alpha.-amylase, i.e. maltose, maltotriose and .alpha.-dextrin, are all further converted to glucose by the action of other enzymes. A different type of amylase, .beta.-amylase, is found in e.g. malt, and hydrolyzes starch to maltose. Starch can in addition be hydrolyzed by the enzyme starch phosphorylase. However, it is believed that only .alpha.-amylase is able to hydrolyze intact starch molecules, while .beta.-amylase and starch phosphorylase are only able to act on the products of .alpha.-amylase hydrolysis. Thus, while .alpha.-amylase hydrolyzes the internal .alpha.-1,4 linkages, .beta.-amylase works only on residues at the non-reducing terminus. .alpha.-Amylase is therefore a necessary enzyme in the hydrolysis of starch and the conversion of starch to e.g. gluose and fructose.
Food is stored in plants in the form of starch, which is converted to sugar when necessary for use as a source of energy for plant growth. Potato tubers are rich in starch, the starch being converted to sugar upon sprouting and, as explained below, under conditions of storage at relatively low temperatures.
It is commonly known that a portion of the starch in stored potatoes becomes hydrolyzed to glucose during storage of the potatoes, and that the glucose is partially converted to fructose. The rate of hydrolysis of starch to the reducing sugars glucose and fructose increases with decreasing temperatures. Thus, potatoes which are stored for a period of time at a temperature of lower than about 7.degree.-8.degree. C. typically have a relatively higher sugar content, while potatoes stored at a temperature of about 8.degree. C. or higher have a relatively lower sugar content. While this might indicate that the problem of a relatively higher content of reducing sugar in potatoes to be used for chips production could be solved by storing the potatoes at about 8.degree. C. or higher, this is not an ideal solution, since potatoes begin to sprout at an unacceptable rate at such relatively high temperatures. Such sprouting may be prevented or counteracted by spraying the potatoes with an antisprouting agent, but from a consumer's point of view, this is not desirable.
It would be advantageous to be able to reduce the amount of sugar in potatoes of a given potato variety. Thus, as explained above, a reduction of the amount of sugar would be advantageous for potatoes used for the production of potato chips, etc. However, it has not previously been possible to achieve a satisfactory regulation of the sugar content in potatoes by traditional plant breeding methods.
The present invention now opens up the possibility of regulating .alpha.-amylase activity by transgenic plant strategies. This and other aspects of the invention will appear from the following description.